Coral bleaching caused by thermal stress is becoming increasingly serious (Hughes et al. 2018). In the last decade, coral bleaching has occurred at significantly higher sea surface temperatures (SSTs) (∼0.5 °C) than in the previous decade, suggesting that thermally susceptible genotypes may have declined and/or adapted (Sully et al. 2019). Zeaxanthin is a carotenoid antioxidant that has been shown to protect endosymbiotic Symbiodiniaceae algae, isolated from coral Galaxea fascicularis, from thermal and light stress (Motone et al. 2020). Coding genes for Zeaxanthin biosynthesis enzymes phytoene desaturase, lycopene beta-cyclase, and beta-carotene 3-hydroxylase were examined in 115 of the 177 total genera in Flavobacteriaceae, and over 50% of these genera (62) had all three enzymes (Table S1). Of note, all 11 strains of coral-associated flavobacteria (from approximately ten genera), including strain R38T, were able to produce zeaxanthin according to HPLC analysis (Table 1). These results indicate that the Flavobacteriaceae family contains important zeaxanthin producers, and corals may benefit from these symbiotic flavobacteria when confronting thermal stress (Motone et al. 2020). However, zeaxanthin in coral (Porites lutea and Acropora sp.) or Symbiodinium was below the limit of HPLC detection (Venn et al. 2006 and this study). Zeaxanthin is an intermediate product of the algal accessory photosynthetic pigments fucoxanthin and peridinin (Dautermann et al. 2020), and may be transformed immediately following synthesis in the coral holobiont. This is suggested by the detection of its precursor, β-carotene, and the downstream product, peridinin, in coral or Symbiodinium (Venn et al. 2006). Although zeaxanthin exchange between bacteria and the endosymbiont Symbiodinium has not been shown, similar effects of pure culture Muricauda sp. GF1 and zeaxanthin supplementation to cultured Symbiodiniaceae conducted by Motone et al. (2020) strongly supports this exchange. Therefore, flavobacterial zeaxanthin may support the biosynthesis of algal accessory photosynthetic pigments in the coral endosymbiont Symbiodinium. Finally, carotenoids can function as antioxidants via epoxidase/de-epoxidase reaction, regardless of the type of carotenoid in the final product (Krinsky 1989; Lacour et al. 2020).
Morphological, physiological, and biochemical analyses
Cells of bacterial strain R38T were gram-negative, non-spore-forming, non-motile, aerobic rods. Cells were usually 0.3-0.5 μm wide and 0.9-2.0 μm long (Fig.1), being narrower than that of L. flavescens KCTC 22160T, S. citrea KCTC 32990T and Fulvibacter tottoriensis MTT-39T, while being wider than that of Mesoflavibacter aestuarii KYW614T (Table 2). Cells of strain R38T could reduce nitrate to nitrogen, L. flavescens KCTC 22160Tand S. citrea KCTC 32990T could only reduce nitrate to nitrite, whereas M. aestuarii KYW614T (Lee et al. 2014) and F. tottoriensis MTT-39T (Khan et al. 2008) could not reduce nitrate. Enzyme characterization of strain R38T using API ZYM strips showed a spectrum similar to that of M. aestuarii KYW614T and F. tottoriensis MTT-39T with the absence of β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, and α-mannosidase (Table 2) (Lee et al. 2014; Yoon et al. 2013). These enzyme results differed from those of L. flavescens KCTC 22160T and S. citrea KCTC 32990T (Table 2). Other characteristics of strain R38T are listed in Table 2 and the species description.
The only respiratory quinone detected in strain R38T was menaquinone 6 (MK-6), similar to L. flavescens KCTC 22160T (Mitra et al. 2009), S. citrea KCTC 32990T (Yoon et al. 2015), F. tottoriensis MTT-39T (Khan et al. 2008), and Mesoflavibacter aestuarii KYW614T (Lee et al. 2014). Strain R38T contained iso-C15 : 0 (42.4 %), iso-C15 : 0ω6c (15.6 %), C16:2 DMA (7.8 %), and C13:1ω3c (5.2 %) as the major cellular fatty acids, this profile was highly similar to that of L. flavescens KCTC 22160T, although the proportion of individual components varied (Table S2). However, the individual fatty acid content of S. citrea KCTC 32990T differed from that of strains R38T and L. flavescens KCTC 22160T (Table S2). The major fatty acids of strain R38T, L. flavescens KCTC 22160T, and S. citrea KCTC 32990T were highly different from those of M. aestuarii KYW614T and F. tottoriensis MTT-39T (Table 2) (Lee et al. 2014; Yoon et al. 2013). The major polar lipids of strain R38T were phosphatidyl-N-methylethanolamine, phosphatidylethanolamine, one unidentified ninhydrin phospholipid, three unidentified ninhydrin-positive lipids, and three unidentified lipids (Supplementary Fig.S1). This polar lipid profile was similar to that of L. flavescens KCTC 22160T, but highly different from that of S. citrea KCTC 32990T, which contains few types of polar lipids (Supplementary Fig.S1). However, strain R38T could still be distinguished from L. flavescens KCTC 22160T by its unidentified ninhydrin phospholipid (Supplementary Fig.S1). Furthermore, no phosphatidyl-N-methylethanolamine has been reported for M. aestuarii KYW614T and F. tottoriensis MTT-39T (Lee et al. 2014; Yoon et al. 2013). Therefore, the polar lipid profile distinguishes strain R38T from any validly published taxon.
Molecular characterization and phylogenetic analysis
A nearly complete 16S rRNA gene sequence (1383 nt) of strain R38T was obtained by Sanger sequencing and deposited in GenBank under accession number MN908337. Global alignment using the EzBioCloud database indicated that the most closely related neighbor of strain R38T is Mesoflavibacter aestuarii KYW614T, with a 16S rRNA gene similarity of 93%. The next most similar members were of Bizionia, Sabulilitoribacter, Gaetbulibacter, and Algibacter genera which showed 92.7-92.9% sequence similarity. However, 16S rRNA gene phylogenetic analysis based on the maximum-likelihood algorithm indicated that strain R38T forms a distinct branch in a stable cluster composed of strain R38T and L. flavescens KCTC 22160T (Fig. 2). Neighbor-joining clustering also supports this tree topology (Supplementary Fig. S2). The close relationship between strain R38T and L. flavescens KCTC 22160T was also represented in the maximum-parsimony analysis, although a robust cluster was not formed (Supplementary Fig.S3).
Genome properties and comparison
The genome sequencing depth of strain R38T was 333×, and the N50 was 750154 bp. A total of 11 contigs were obtained, the obtained genome size was 3.94 Mb, and the genomic DNA G+C content was 33.2 mol%. The genome sequencing depth of L. flavescens KCTC 22160T was 154×, the N50 was 1032064 bp, a total of 9 contigs were obtained, the obtained genome size was 4.21 Mb, and the genomic DNA G+C content was 40.9 mol%. The genome sequencing depth of S. citrea KCTC 32990T was 155×, the N50 was 637254 bp, a total of 17 contigs were obtained, the obtained genome size was 4.15 Mb, and the genomic DNA G+C content was 36.3 mol%. The genomes of closely related type strains were 3.05-5.03 MB, with G+C content of 33.2-55.3 mol% (Supplementary Table S3). Thus, strain R38T has the lowest genomic G+C content (33.2 mol%). The complete 16S rRNA gene of strain R38T obtained by genome sequencing was 1508 nt, and showed two nucleotide differences compared to the sequence obtained by Sanger sequencing. Meanwhile, the result obtained by Sanger sequencing indicated there were at least two copies of the 16S rRNA gene in cells of strain R38T. The ANI of strain R38T to L. flavescens KCTC 22160T, S. citrea KCTC 32990T, and M. aestuarii KYW614T were 72.5%, 69.6%, and 70.2%, respectively. The AAI of strain R38T to L. flavescens KCTC 22160T was 74.6%, and for other type strains, the indices were lower than 70%. Similar to the 16S rRNA gene-based phylogenetic results, phylogenomic analysis based on 92 genes also indicated that strain R38T forms a distinct branch in a stable cluster composed of strain R38T and L. flavescens KCTC 22160T (Supplementary Fig.S4).
Approximately 37 families of carbohydrate-active enzymes were detected in strain R38T, while in closely related type strains this number was between 39-100 (Supplementary Table S3). A limited quantity of glycoside hydrolases (16 vs 20-63) indicates that strain R38T is weak in carbohydrate utilization (Supplementary Table S3). Approximately 71 families of peptidases were detected in strain R38T. This quantity is higher than in most of the closely related type strains (Supplementary Table S3), indicating that strain R38T is versatile in protein utilization. This carbohydrate and protein utilization pattern might have resulted from long-term bacteria-animal association.
Based on phylogenetic analyses, strain R38T was found to be associated with the family Flavobacteriaceae. The ANI of strain R38T to closely related type strains (≤72.5%) indicates that strain R38T belongs to a novel species (Chun et al. 2018), and both biochemical and chemotaxonomic characteristics (Table 2) support this species-level assignment. Furthermore, the low 16S rRNA gene similarities (≤93%) of strain R38T to closely related type strains indicate that strain R38T represents a new genus (Yarza et al. 2014), which is also supported by the differences in polar lipid profile (Table 2 & Supplementary Fig.S1). Therefore, strain R38T represents a new species in a new genus under the family Flavobacteriaceae, for which Prasinibacter corallicola gen. nov., sp. nov. is proposed.
Description of Prasinibacter gen. nov.
Prasinibacter (Pra.si.ni.bac′ter. L. masc. adj. prasinus, yellowish-green; N.L. masc. bacter, rod: N.L. masc. n. Parisinibacter, a translucent yellowish green rod.)
Cells are gram-negative, non-spore-forming, non-motile, aerobic rods. Catalase- and oxidase-positive. Nitrate is reduced to nitrogen. The only menaquinone is MK-6. The major polar lipids are phosphatidyl-N-methylethanolamine, phosphatidylethanolamine, one unidentified ninhydrin phospholipid, three unidentified ninhydrin-positive lipids, and three unidentified lipids.
The type species is Prasinibacter corallicola. Member of the family Flavobacteriaceae.
Description of Prasibacter corallicola sp. nov.
Prasinibacter corallicola (co.ral.li.co′la. L. neut. n. corallum, coral; L. masc. suff. -cola, inhabitant dweller; N.L. n. corallicola, coral-dweller)
The description is as for the genus with the following additional properties: Cells are usually 0.3-0.5 μm wide and 0.9-2.0 μm long. Colonies are yellow-green, circular, and smooth on marine agar 2216. Cells can grow at 15-33 °C (optimum 25-30°C), pH 5-10 (optimum 7-8) in 3-6% (w/v) NaCl (optimum 3-4%) in R2A liquid medium. Zeaxanthin is produced. Production of H2S does not occur. Starch is hydrolyzed. In the API 20NE test, nitrate reduction and protease are positive. In the API ZYM test, alkaline phosphatase, esterase (C4), esterase lipase (C8) and (C14), leucine arylamidase, valine arylamidase, cysteine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, naphthol-AS-BI phosphohydrolase, and α-galactosidase are positive. In the Biolog Gen III microplate, dextrin, D-maltose, α-D-glucose, D-mannose, L-alanine, L-glutamic acid, L-histidine, α-keto-glutaric acid, L-malic acid, bromo-succinic acid, Tween 40, acetoacetic acid, and acetic acid are oxidized. The major fatty acids are iso-C15 : 0, iso-C15 : 0ω6c, C16:2 DMA, and C13:1ω3c. The genomic DNA G+C ratio is 33.2 mol%.
The type strain, R38T (=MCCC 1K03889T=KCTC 72444T) was isolated from stony coral Porites lutea collected from Weizhou Island in the Beibu Gulf, China. The GenBank accession number of the 16S rRNA gene sequence of the type strain is MN908337.